FcγR介导SOCS2/3信号转导调控巨噬细胞极化在胃癌转移中的作用及机制

The polarization of M2 Macrophage plays an important role in promoting the tumor invasion and metastasis. The latest studies have shown that B cells can promote M2 macrophage polarization, however the exact mechanism is not yet known. Our previous studies showed that B cells co-existed with the polarized M2 macrophages in the gastric cancer microenvironment, whitch promote the metastasis of gastric cancer. Therefore we propose that the B cells may induce the macrophage polarization through the SOCS2/3 signaling pathway mediated by FcγR in the gastric cancer microenvironment. This project is intended to explore the role of B cell on macrophage polarization with in vitro/vivo experiments and co-culture model. Using in vitro over-expression/silencing SOCS proteins model and SOCS knockout mice experiments combined with mass spectrometry, we will determine the key regulatory role of FcγR-mediated SOCS2/3 signaling in macrophage polarization and dynamic observation of gastric cancer metastasis model with in vivo imaging technology. By Transwell and ECM adhesion assay, we will explore the the role and mechanism of the FcγR polarizated macrophage cells on gastric cancer invasion and migration. The study is expected to clarify the new mechanism of M2 macrophage polarization in the tumor microenvironment and the exact role involved in the metastasis of gastric cancer, and to provide new theoretical and experimental basis to clear the tumor microenvironment in gastric cancer metastasis.

巨噬细胞向M2亚型极化对肿瘤的侵袭和转移起重要的促进作用，最新的研究显示B细胞可促进M2巨噬细胞的极化，但具体机制未知。我们的前期研究显示胃癌微环境中B细胞和极化的M2巨噬细胞共存，且促进胃癌的转移。因此提出假设胃癌微环境中B细胞可能通过FcγR介导SOCS2/3信号调控MФ极化而促进转移。本项目拟应用体内体外实验和共培养模型研究B细胞对巨噬细胞极化作用；利用体外过表达/沉默SOCS蛋白及SOCS敲除小鼠体内实验结合质谱技术确定SOCS2/3在FcγR介导巨噬细胞极化中的关键调控作用及上下游信号分子；通过Transwell，ECM黏附等方法研究FcγR极化的巨噬细胞对胃癌侵袭迁移的作用和机制；利用胃癌转移模型和活体成像技术动态观察胃癌荷瘤小鼠的肿瘤转移情况。该研究有望阐明肿瘤微环境中M2巨噬细胞极化的新机制及参与胃癌转移的确切作用，为明确肿瘤微环境在胃癌转移作用提供新的理论和实验依据。

本课题组前期研究证实胃癌组织肿瘤相关巨噬细胞（TAM）增多，促进胃癌侵袭转移。但目前对胃癌组织中TAM浸润特征及相关极化调节机制尚不明确。因此，在前期临床肿瘤样本检测和功能实验基础上，结合体内外模型对胃癌组织巨噬细胞（MФ）极化调节机制提出了新的研究思路和模式，并进一步深入探讨了MФ极性分化对胃癌转移作用机制。实验发现肿瘤微环境内及体外极化TAM高分泌TGF-β，并介导肿瘤细胞迁移。同时检测发现胃癌组织中存在的大量TAM与肿瘤相关成纤维细胞（α-SMA+ CAF）数量和分布呈正相关。体外分离CAF以及M2 TAM诱导转化CAF均可促进胃癌细胞转移潜能。对FcγR通路SOCS2/3分子体外过表达研究发现，SOCS3过表达诱导M1型MФ极化，SOCS2过表达诱导M2极化。 FcrR活化MФ促进腹腔种植瘤生长和MFC细胞肺转移。Transwell实验明确M2 TAM在体外显著增强胃癌细胞迁移及侵袭。蛋白印迹实验表明胃癌细胞与M2 TAM共培养后，EMT相关蛋白slug、snail等转录调控因子表达增加。细胞免疫荧光染色显示胃癌细胞与M2 TAM共培养后细胞骨架发生改变。流式细胞术检测证实，与脾脏中MФ相比，小鼠肿瘤组织中MФ也大多为M2 TAM（CD11b+/F480+/CD206+）。体外分离M2 TAM培养上清外泌体，发现其可被胃癌细胞体外摄取。阻断M2 TAM外泌体分泌可减弱其促迁移侵袭作用。此外，与M2 TAM共培养后，胃癌细胞MFC肺部转移能力及腹腔种植转移能力显著增强。且发现这一功能改变与PI3K-AKT-mTOR通路密切相关。本项目顺利完成有助于深入理解胃癌微环境中，M2 TAM结合CAF在分泌TGF-β和Exosome作用下，以间接作用方式通过EMT途径和PI3K-AKT-mTOR信号，促进胃癌细胞转移新机制。